Flow cytometers are frequently used for the analysis of particles such as cells or beads in a number of different applications. The system allow for determination of both particle morphology and evaluation of particle features by detection of optical labels. The ability to distinguish multiple particles sizes and colors allows multiplex application providing higher capacity of this technology to obtain information from analyzed targets.
Particle as used herein means any discrete target that may be optically analyzed, enumerated or sorted by a flow cytometer. This group includes cells, cell fragments and beads.
In flow cytometer systems liquid containing target particles are fed from a container into a flow cell. The flow cells separates particles into a stream of individual particles that flow past a detection location. The particles may flow as individual droplets, but to reduce optical noise from refraction it is often preferred to have the particle stream flow through a cuvette where particles in the flow stream are analyzed. At the detection location a beam of focused illumination light (often a laser beam) illuminates the passing particles.
Light scattered by the passing particles is detected by forward and side scatter detectors allowing determination of particle morphology. Light emitted from particles is collected and transmitted to detection optics. The particle (generally a cell or bead) may be labeled with one or more dye having a characteristic excitation and fluorescent emission wavelength. The dye may be conjugated to a binding agent (e.g. a monoclonal antibody) allowing targeting of specific antigen associated with the bead or cell. Light beam splitters separate the collected light into component wavelengths. These beams are directed through a bandpass filter to a light detector (e.g. photomultiplier tube). A specific wavelength associated with each dye is individually detected by one detector.
The particle, after passing through a flow cell may pass into a sorting block for cell sorting operations. The flow cell nozzle breaks the flow stream into individual droplets, each droplet potentially containing one or more particle. If the particle is to be collected, the droplet is given a charge by a charging system. The droplets then fall in a stream past charged deflection plates in the sort block. Charged droplets will be deflected in one of two directions, depending on the charge applied to the droplet. The uncharged droplets are not deflected and may be collected as unsorted particles. The charged droplets are deflected and exit the sort block to collection containers. If the particles are cells, the sorted cells may then be used for additional procedures. By applying different levels of positive or negative charge a single sort procedure may sort the flow stream into four containers.
There are a number of potential problems with cell sorters. First, if the sorter malfunctions particles could be sprayed as an aerosol through the sort block. The sorted particles in containers would be contaminated and need to be resorted. Such a malfunction could include a block in the flow cell resulting in the flow stream being aerosoled into a fine mist through the sort block. In addition, pressure changes could result in spraying from the sort nozzle. Alternatively, error in the charging system could also misdirect particles. This spraying malfunction would result in the contamination of sorted particles, potentially wasting hours of sorting time. The repeated sorting of cells may also negatively affect cell vitality and viability.
A second potential problem is the aerosols generated by the sort nozzle. The particles are sorted into droplets that fall to the container. The particles are of sufficient size that the flow direction and the droplet weight act to draw the droplet to the sort containers or into a collector for unsorted cells. However in the formation of droplets some fluid may form an aerosol in the sorting block. The aerosol is composed of a fine mist that can be suspended in the air in the sort block. An aerosol could escape the sort block if it is not airtight and expose the user to the aerosol. This can be hazardous, especially given that sorting of blood cells is a common flow cytometry procedure. A virus, such as HIV or the Hepatitis virus, could escape the sort block in an aerosol. The flow cytometer user could then be exposed to this virus. If radioactive agents are used, exposure of the user to radioactive agents would also pose hazard from aerosols generated.
The potential hazards are evaluated in Cytometry 28:99-117(1997). The recommendations for safety noted in this publication include decontamination of an instrument with a disinfecting agent following use, a vacuum containment system on the sorter to remove aerosols (while ensuring that air turbulence is not created in the sort stream), removal of unsorted cells to a waste stream, and enclosure of the sort system in a housing to prevent aerosol escape. This housing has generally contained the flow cell and sort block, containing all elements in an enclosed chamber to prevent aerosol escape. The housing may then be evacuated with a vacuum following a sort process, ensuring that any aerosol generated is evacuated through a filter cartridge.
Most flow cytometers rely on manual shut off to prevent system error. However, in the time required to shut off the flow system, a sorting process could be ruined. If a containment housing is used, the housing must be evacuated and sterilized and the sort repeated.
U.S. Pat. No. 5,776,781 discloses a system for isolation of flow cytometer elements to enclose the system and prevent the escape of aerosols. In addition, the system has a component to sterilize the enclosed area using a chemical or UV light sterilizer. The system maintains positive pressure within the isolation chamber to maintain sterility and ensure that particles from outside the isolation chamber do not gain entrance. The isolation chamber encloses the flow cell, droplet generator, charging system, deflecting system and cell collector. As in other systems, a significant volume of space must be enclosed, adding bulk to the system. Additional lab space is required for such large systems.
PCT document WO 01/85088 A1 discloses a safety cabinet for use with a flow cytometer. The flow cytometer is enclosed within the cabinet. A blower/filtration means removes and filters air from the cabinet, maintaining negative pressure within the cabinet.
U.S. Pat. No. 6,248,590 discloses a system in which a flow interrupter is brought into the flow stream to divert the entire flow stream in instances where an optical stream evaluation system determines that some required parameter is not satisfied. The stream is then shunted to waste by a deflector or gutter introduced into the flow area.
Past devices have achieved a safer containment at the cost of system bulk. The aspirators employed to maintain a negative air pressure within the sorting block would have to provide enough pressure to evacuate the contained area which may be a sizeable volume. In addition a robust system for protecting cells that have been sorted from sprays resulting from malfunction is not presently available.
It is an object of the present invention to provide a flow cytometer sort block that provides a means for detecting system error and collecting the entire flow stream and shunting the flow stream from the collection containers. This would prevent the collected cells or beads from being contaminated.
It is a further object of the invention to provide a sort block that prevents aerosol escape.
It is a further object of the invention to provide a means for introducing negative pressure within the sort block.